Control of receptor localization to the plasma membrane is central to normal cell function, and dysregulation is the underlying cause for diseases as diverse as atherosclerosis, diabetes and cancer. Receptor levels on the plasma membrane are dependent upon the rates of internalization and recycling back to the cell surface. We and others have recently identified a novel Eps15 homology (EH)-domain containing protein, EHD1, as a critical component of the endocytic recycling pathway. The physiologic importance of EHD-mediated function has been clearly demonstrated, as loss of EHD1 function leads to impaired recycling of many critical receptors. The long- term goals of this project are to understand the fundamental mechanisms controlling intracellular trafficking and transport of proteins to the plasma membrane, with emphasis on understanding the mode by which EHD structure impacts its function. The central hypothesis of this proposal is that the electrostatic surface charge of the EH-domain of EHD1 (EH-1) and its C-terminal EHD paralogs is responsible for the selectivity of interactions with NPF-containing protein partners and phosphoinositides, and that these interactions are important for the regulation of endocytic transport. We provide compelling evidence for our hypothesis based on our strong preliminary data that EH-1 selectively interacts with proteins containing NPF motifs followed by a cluster of negatively charged residues. In addition, our new preliminary data also reflect the functional importance of the EHD/phosphoinositide interactions by demonstrating that EH-1 is capable of interacting directly with phosphatidylinositol moieties, and that this interaction is required to allow localization of EHD1 to tubular and vesicular membrane structures. Our Specific Aims for the proposal are: 1) To identify the molecular and structural basis for the selectivity of C-terminal EH-domains for specific NPF-containing proteins, and 2) To elucidate the specific phosphoinositide(s) that are enriched/comprise EHD tubular membranes and determine the significance of EHD/phosphoinositide binding in the regulation of endocytic trafficking. The knowledge to be obtained from this study will provide critical new information on the mode by which the C-terminal EHD proteins associate with endosomal membranes and coordinate control of recycling. This will lead to a significantly enhanced understanding of the endocytic mechanisms that regulate receptor recycling, and will have an important bearing on health and disease.